Kallikrein transduced mesenchymal stem cells protect against anti-GBM disease and lupus nephritis by ameliorating inflammation and oxidative stress

Abstract

Previously we have shown that kallikreins (klks) play a renoprotective role in nephrotoxic serum induced nephritis. In this study, we have used mesenchymal stem cells (MSCs) as vehicles to deliver klks into the injured kidneys and have measured their therapeutic effect on experimental antibody induced nephritis and lupus nephritis. Human KLK-1 (hKLK1) gene was transduced into murine MSCs using a retroviral vector to generate a stable cell line, hKLK1-MSC, expressing high levels of hKLK1. 129/svj mice subjected to anti-GBM induced nephritis were transplanted with 10(6) hKLK1-MSCs and hKLK1 expression was confirmed in the kidneys. Compared with vector-MSCs injected mice, the hKLK1-MSCs treated mice showed significantly reduced proteinuria, blood urea nitrogen (BUN) and ameliorated renal pathology. Using the same strategy, we treated lupus-prone B6.Sle1.Sle3 bicongenic mice with hKLK1-MSCs and demonstrated that hKLK1-MSCs delivery also attenuated lupus nephritis. Mechanistically, hKLK1-MSCs reduced macrophage and T-lymphocyte infiltration into the kidney by suppressing the expression of inflammation cytokines. Moreover, hKLK1 transduced MSCs were more resistant to oxidative stress-induced apoptosis. These findings advance genetically modified MSCs as potential gene delivery tools for targeting therapeutic agents to the kidneys in order to modulate inflammation and oxidative stress in lupus nephritis.

Figure 1. hKLK1-MSCs with constitutive expression of hKLK1.

(A, B) Detection of hKLK1 expression in MSCs by QPCR (A) and western blot (B). (C) Immunophenotypic characterization of MSCs. In vitro cultured hKLK1-MSCs were stained with antibodies against different cell surface antigens and analyzed by flow cytometry. Gray-filled histograms show flow cytometric analysis of cells stained with isotype control whereas blue thick lines display cells incubated with specific antibodies: Sca-1, CD44, CD29 are positive MSC markers, and CD45, CD11b, CD34 are negative MSC markers.

Figure 2. hKLK1 expression in the kidney of B6.Sle1.Sle3 mice receiving hKLK1-MSCs.

(A) hKLK1 mRNA expression was detected by QPCR in the kidney of B6.Sle1.Sle3 mice injected with hKLK1-MSCs, but not in vector-MSCs or PBS-injected B6.Sle1.Sle3 mice (p<0.01). (B) hKLK1 protein expression was detected by western blot in the kidneys of hKLK1-MSCs injected B6.Sle1.Sle3 mice, but not in vector-injected B6.Sle1.Sle3 mice. Three mice were examined for each group.

Figure 3. hKLK1-MSCs transfer suppressed anti-GBM induced nephritis in 129/svj mice.

129/svj mice were challenged with anti-GBM antibody on day 0 and then treated with hKLK1-MSCs, vector-MSCs or PBS (sham control). Proteinuria (A) and serum BUN (B) were measured on day 0, day 14 and day 21. The values represent the average of 7 mice in each group. *p<0.05, **p<0.01. (C) Renal sections were evaluated for glomerulonephritis (GN) under light microscopy on day 21. (D–F) Shown are periodic acid schiff-stained, formalin-fixed, paraffin-embedded renal sections from 129/svj mice treated with PBS (D), vector-MSCs (E), or hKLK1-MSCs (F). Images are representative of sections from at least 6 mice in each study group (Original magnification 400×).

Figure 4. hKLK1-MSCs transfer ameliorated spontaneous lupus nephritis in B6.Sle1.Sle3 mice.

8-month old female B6.Sle1.Sle3 mice were treated with 10⁶ vector-MSCs, the same numbers of hKLK1-MSCs, or equal volume of PBS, respectively, via tail vein injection. Proteinuria (A) and serum BUN (B) were measured on day 0 (before treatment) and day 28 (after treatment). Each bar represents the average of 6 mice in each group. (C) Kidneys were collected on day 28 and renal GN score was evaluated under light microscopy. (D–F) Shown are periodic acid schiff-stained, formalin-fixed, paraffin-embedded renal sections from B6.Sle1.Sle3 mice treated with PBS (D), vector-MSCs (E), or hKLK1-MSCs (F). Images are representative of sections from at least 5 mice in each study group (Original magnification 400×).

Figure 5. hKLK1-MSCs were resistant to H₂O₂ induced apoptosis and hKLK1-MSCs transplantation reduced renal cell apoptosis in B6.Sle1.Sle3 mice.

(A–C) Detection of apoptosis by TUNEL staining of in vitro cultured unmodified MSCs (A), vector-transduce MSCs (B) or hKLK1-transduced MSCs (C) treated with 0.5 mM H₂O₂ for 6 hr. The apoptotic cells appear as intense fluorescent signals (arrow). (D) Plotted are the percentages of apoptotic cells in each group. (E–G) Detection of apoptotic cells in the kidneys of B6.Sle1.Sle3 mice treated with PBS (E), vector-MSCs (F) or hKLK1-MSCs (G) for 28 days by TUNEL staining. Apoptotic cells appear as dark brown (arrow). (H) Plotted are the percentages of apoptotic cells in the kidney. Data shown are representative of data from 5 mice in each group.

Figure 6. hKLK1-MSC transfer reduced renal macrophage and T-lymphocyte infiltration.

Immunohistochemical staining on formalin-fixed, paraffin-embedded kidney tissue sections. Kidney sections from B6.Sle1.Sle3 mice treated with PBS (A, E), vector-MSCs (B, F) or hKLK1-MSCs (C, G) were stained with macrophage specific anti-Iba1 antibody (1∶800 dilution), or T-lymphocyte specific anti-CD3 antibody (1∶300 dilution). D and H are the percentage of Iba1 positive cells (D) and CD3 positive cells (H). Data shown are representative of 6 mice in each group (Original magnification 400×).

Figure 7. hKLK1-MSCs transfer down-regulated the expression of inflammatory cytokines and apoptosis associated cytokines in mouse kidney.

The expression of 13 genes representing proinflammatory cytokines, chemokines and apoptotic factors in the kidneys were measured by QPCR in B6.Sle1.Sle3 mice treated with PBS, hKLK1-MSCs or vector-MSCs. Total renal RNA was extracted 28 days after treatment. Taqman assay was performed using an ABI 7900HT real-time PCR system. RQ (relative quantity) represents the mean of 5 samples per group. Error bars denote SD.

Figure 8. Levels of inflammatory cytokines in mouse serum measured using a Luminex multiplex Assay.

8-month-old B6.Sle1.Sle3 mice were injected with PBS, 10⁶ hKLK1-MSCs or vector-MSCs and sera were collected on day 28 after treatment. IL-10 expression was increased, while IL-1β, IL-2, IL-6, IL-12, IL-13, IP-10, KC, MIG, MIP-1α, TNF-α and VEGF were decreased in the sera of hKLK1-MSCs treated mice compared with both PBS treated mice and vector-MSCs treated mice. Each bar represents the mean of 3 mice. Error bars denote SD.